Species identification of cell lines and detection of cross-contamination are crucial for scientific research accuracy and reproducibility. Whereas short tandem repeat profiling offers a solution for a limited number of species, primarily human and mouse, the standard method for species identification of cell lines is enzyme polymorphism. Isoezymology, however, has its own drawbacks; it is cumbersome and the data interpretation is often difficult. Furthermore, the detection sensitivity for cross-contamination is low; it requires large amounts of the contaminant present and cross-contamination within closely related species may go undetected. In this paper, we describe a two-pronged molecular approach that addresses these issues by targeting the mitochondrial genome. First, we developed a multiplex PCR-based assay to rapidly identify the most common cell culture species and quickly detect cross-contaminations among these species. Second, for speciation and identification of a wider variety of cell lines, we amplified and sequenced a 648-bp region, often described as the "barcode region" by using a universal primer mix targeted at conserved sequences of the cytochrome C oxidase I gene (COI). This method was challenged with a panel of 67 cell lines from 45 diverse species. Implementation of these assays will accurately determine the species of cell lines and will reduce the problems of misidentification and cross-contamination that plague research efforts.
The role of cell authentication in biomedical science has received considerable attention, especially within the past decade. This quality control attribute is now beginning to be given the emphasis it deserves by granting agencies and by scientific journals. Short tandem repeat (STR) profiling, one of a few DNA profiling technologies now available, is being proposed for routine identification (authentication) of human cell lines, stem cells, and tissues. The advantage of this technique over methods such as isoenzyme analysis, karyotyping, human leukocyte antigen typing, etc., is that STR profiling can establish identity to the individual level, provided that the appropriate number and types of loci are evaluated. To best employ this technology, a standardized protocol and a data-driven, quality-controlled, and publically searchable database will be necessary. This public STR database (currently under development) will enable investigators to rapidly authenticate human-based cultures to the individual from whom the cells were sourced. Use of similar approaches for non-human animal cells will require developing other suitable loci sets. While implementing STR analysis on a more routine basis should significantly reduce the frequency of cell misidentification, additional technologies may be needed as part of an overall authentication paradigm. For instance, isoenzyme analysis, PCR-based DNA amplification, and sequence-based barcoding methods enable rapid confirmation of a cell line’s species of origin while screening against cross-contaminations, especially when the cells present are not recognized by the species-specific STR method. Karyotyping may also be needed as a supporting tool during establishment of an STR database. Finally, good cell culture practices must always remain a major component of any effort to reduce the frequency of cell misidentification.
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